148 CHEMISTRY & BIODIVERSITY Vol.

12 (2015)

Two New Hydronaphthoquinones from Sinningia aggregata (Gesneriaceae)

and Cytotoxic Activity of Aggregatin D
by Maria Helena Verdan* a ), Lauro Mera de Souza b ), Joao Ernesto de Carvalho c ),
Debora B. Vendramini Costa c ), Marcos Jose Salvador d ), Andersson Barison a ), and
Maria Elida Alves Stefanello a )
a
) Department of Chemistry, Federal University of Parana, Curitiba, PR, 81531-980, Brazil
(phone: 55-41-33613177; fax: 55-41-33613186; e-mail: mhelenaverdan@gmail.com)
b
) Department of Biochemistry and Molecular Biology, Federal University of Parana, Curitiba, PR,
81531-980, Brazil
c
) CPQBA-UNICAMP, State University of Campinas, Campinas, SP, 13083-970, Brazil
d
) Department of Plant Biology, Institute of Biology/Faculty of Pharmaceutical Sciences, PPG-BTPB,
University of Campinas, Campinas, SP, 13083-970, Brazil

Two new hydronaphthoquinones, aggregatins E and F (1 and 2, resp.) were isolated from the tubers
of Sinningia aggregata (Ker-Gawl.) Wiehler (Gesneriaceae), along with twelve known compounds
aggregatin D (3), tectoquinone (4), 1-hydroxy-2-methylanthraquinone (5), icosyl ferulate (6), pustuline
(7), 1,6-dihydroxy-2-methylanthranquinone (8), 6-hydroxy-2-methylanthraquinone (9), 7-hydroxy-2-
methylanthraquinone (10), tyrosol (11), halleridone (12), calceolarioside B (13), and cornoside (14). All
compounds were identified by analysis of spectroscopic and spectrometric data. Compounds 3, 4, and 10
had already been reported in this species. Compounds 2 and 3 were evaluated against several tumor cell
lines, but only 3 exhibited activities against UACC-62, 786-0 and OVCAR-3 cell lines, with IC50 values of
12.3, 12.8 and 0.3 mg/ml, respectively, without toxic effects on non-cancer cell line HaCat (human
keratinocyte).

Introduction. The genus Sinningia (Gesneriaceae) is neotropical and comprises 68

species, widely distributed in Brazil [1]. S. aggregata (Ker-Gawl. ) Wiehler is an
annual herbaceous plant that reaches a height of 1.5 m, with perennial tubers and aerial
parts covered by an aromatic resin. It is native of Brazil and Paraguay [2]. We have
already reported the composition of leaf essential oil [3] and the isolation of e-lactones
(aggregatins A C), a hydronaphthoquinone (aggregatin D), and three anthraqui-
nones [4]. Herein, we describe the isolation and structure elucidation of two new
hydronaphthoquinones, 1 and 2, along with twelve known compounds that were
identified as aggregatin D (3) [4], tectoquinone (4) [5], 1-hydroxy-2-methylanthra-
quinone (5) [6], icosyl ferulate (6) [7], pustuline (7) [8], 1,6-dihydroxy-2-methylan-
thraquinone (8) [9], 6-hydroxy-2-methylanthraquinone (9) [10], 7-hydroxy-2-methyl-
anthraquinone (10) [11], tyrosol (11) [12], halleridone (12) [13], calceolarioside B (13)
[14], and cornoside (14) [15]. Among known compounds, only 3, 4, and 10 had been
previously found in S. aggregata [4]. In addition, the cytotoxicities of 2 and 3 were
evaluated.

 2015 Verlag Helvetica Chimica Acta AG, Zrich

 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 149

Results and Discussion. Aggregatin E (1) was obtained as yellow oil with a
molecular formula C15H14O4 (deduced from HR-ESI-MS and NMR data), consistent
with nine degrees of unsaturation. The 1H-NMR spectrum (Table 1) showed signals
of four aromatic H-atoms, as a spin system of a 1,2-disubstituted benzene (d(H)
7.54 8.10). Additionaly, signals of three olefinic H-atoms (d(H) 6.44 and 6.47 (2d),
and 6.15 (s)), two O-bearing CH2 H-atoms (d(H) 3.84 and 4.00 (2d)), and a Me group
(d(H) 1.71 (s)). These data are very similar to those of aggregatin D (3) previously
reported in S. aggregata [4], suggesting the same skeleton, but with replacement of the
prenyl group at C(6) by a H-atom. Analysis of the HSQC correlations and HMBCs
confirmed the structure of 1 as depicted. In particular, cross-peaks observed in the
HMBC spectrum, Me(12)/C(2), C(3) and C(4), and CH2(2)/C(4) and C(11b),
established the dihydro-methyl-oxepine moiety. The correlations HC(8)/C(7), and
HC(6)/C(5) and C(11b) led to the identification of the hydronaphthoquinone moiety
(Table 1).
Aggregatin F (2) was isolated as yellow oil. Its molecular formula was deduced as
C15H14O5 from HR-ESI-MS and NMR data, compatible with nine degrees of
unsaturation. The 1H- and 13C-NMR spectra (Table 1) were similar to those of 1,
indicating the same basic structure. However, the 1H-NMR spectrum of 2 showed a
typical signal of a OH group with an intramolecular H-bond (d(H) 12.30) and a spin
system, corresponding to three aromatic H-atoms, characteristic of a 1,2,3-trisubsti-
 150 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

Table. 1H- and 13C-NMR Data of 1 and 2 (in CDCl3 ; 400 and 100 MHz, resp.). d in ppm, J in Hz.

Position 1 2
d( H) d(C ) HMBC d(H ) d(C ) HMBC
(H ! C) ( H ! C)
2 3.84 (d, J 6.4, 1 H ), 74.8 (t) 3, 4, 12 3.84 (d, J 6.6, 1 H ), 74.4 (t) 3, 4, 12
4.00 (d, J 6.4, 1 H ) 4, 11b 3.97 (d, J 6.6, 1 H ) 4, 11b
3 81.0 (s) 89.9 (s)
4 6.47 (d, J 9.5, 1 H ) 141.1 (d) 3, 5a, 11b 6.51 (d, J 9.4, 1 H ) 141.8 (d) 3, 5a
5 6.44 (d, J 9.5, 1 H ) 126.3 (d) 3, 5a 6.43 (d, J 9.4, 1 H ) 125.7 (d) 3, 5a, 11b
5a 149.3 (s) 150.7 (s)
6 6.15 (s, 1 H) 122.6 (d) 5, 7a, 11b 6.10 (s, 1 H ) 121.7 (d) 5, 7a, 11b
7 184.5 (s) 189.9 (s)
7a 131.8 (s) 115.3 (s)
8 8.10 (dd, J 7.8, 1.4, 1 H ) 126.3 (d) 7, 10, 11a 161.5 (s)
9 7.54 (ddd, J 7.8, 7.6, 1.3, 1 H ) 130.1 (d) 7a, 11 7.04 (dd, J 8.3, 1.1, 1 H ) 119.2 (d) 7a, 11
10 7.66 (ddd, J 7.8, 7.6, 1.4, 1 H ) 133.2 (d) 8, 11a 7.55 (dd, J 8.3, 7.7, 1 H) 135.7 (d) 8, 11a
11 7.77 (dd, J 7.8, 1.3, 1 H ) 126.4 (d) 7a, 9, 11b 7.26 (dd, J 7.7, 1.1, 1 H ) 117.5 (d) 7a, 9, 11b
11a 137.6 (s) 138.0 (s)
11b 99.2 (s) 99.0 (s)
12 1.71 (s, 3 H ) 19.7 (q) 2, 3, 4 1.70 (s, 3 H ) 19.6 (q) 2, 3, 4
HOC(8) 12.30 (s, 1 H ) 7a, 8, 9

tuted benzene ring, indicating the presence of a OH group at C(8). The structure was
confirmed by HSQC correlations and HMBCs (Table 1).
The relative configurations of 1 and 2 were assigned by comparison of NMR data
with those previously described for compound 3 [4]. However, the signs of the optical
rotation and CD curves for 1 and 2 (Fig.) were opposites of those of 3, suggesting

Figure. CD Spectra of compounds 1, 2, and 3

 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 151

inverted absolute configuration. The absolute configuration of aggregatin D was

predicted by DFT calculations as (11bR,3S) [4]. Therefore, according to experimental
data, the configuration of aggregatins E and F (1 and 2, resp.) was assigned as
(11bS,3R).
Compounds 2 and 3 were assayed against UACC-62 (melanoma), MCF-7 (breast),
NCI-ADR/RES (drug-resistent ovarian), 786 0 (kidney), NCI-H460 (lung, no small
cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), and K562 (leukemia)
cancer cell lines, and also no cancer cell line HaCat (human keratinocyte). Com-
pound 2 was considered inactive, since it showed an IC50 value > 15 mg/ml for all can-
cer cell lines tested. Compound 3 exhibited cytotoxic activities against UACC-62,
786 0 and OVCAR-3 cell lines, with IC50 values of 12.3  1.2, 12.8  1.1, and 0.3 
0.0 mg/ml, respectively. Both compounds did not show toxic effects on HaCat cells
(IC50 > 100 mg/ml).

The authors are grateful to CNPq and FAPESP for financial support, and to Clarisse B. Poliquesi
(Museu Botanico Municipal de Curitiba) for plant identification.

Experimental Part
General. TLC and prep. TLC (PTLC): precoated silica-gel GF254 glass plates (Macherey-Nagel,
Germany). Column chromatography (CC): silica gel (SiO2 , 230 400 mesh; Merck, Germany). Optical
rotations: Jasco P-2000 Polarimeter. UV Spectra: Shimadzu UV-2450 spectrophotometer. CD Spectra:
Jasco J-815 CD Spectrometer; in CHCl3 . NMR spectra: Bruker Avance 400 (at 400 (1H) and 100
(13C) MHz) spectrometer, in CDCl3 or CD3OD, with TMS as the internal reference (d 0 ppm). High
resolution (HR) MS: Thermo Scientific LTQ-Orbitrap XL, with electrospray ionization source (ESI).
The samples were dissolved in MeOH/H2O (7 : 3, v/v) containing 2mm LiCl and directly infused into ESI
source at a flow rate of 5 ml/min.
Plant Material. Tubers of S. aggregata were collected in Tibagi, Parana State, Brazil, in May 2011, and
identified by Clarisse B. Poliquesi. A voucher specimen (No. 290738) was deposited with the herbarium
of the Museu Botanico Municipal (MBM), Curitiba, Brazil.
Extraction and Isolation. Dried and powdered tubers (340 g) were extracted at r.t. with hexane,
followed by AcOEt (1.5 l for 24 h, three times each solvent), to give hexane and AcOEt extracts, after
solvent removal under reduced pressure. The hexane extract (1.5 g) was subjected to CC (hexane/
acetone 1 : 0 to 0 : 1) to give seven fractions, Frs. 1A 7A. Fr. 3A (36.7 mg) was purified by PTLC (hexane/
acetone 8 : 2) to yield 1 (3.2 mg), 2 (2.2 mg) and 3 (9.5 mg). The AcOEt extract (1.9 g) was submitted to
CC (CH2Cl2/AcOEt 1 : 0 to 0 : 1) to give ten fractions, 1B 10B. Fr. 3B (24.8 mg) furnished 4 (3.7 mg) and
5 (2.1 mg) after PTLC (hexane/CH2Cl2 2 : 1). Fr. 5B (17.1 mg) yielded 3 (5.6 mg) and 6 (4.1 mg) after
PTLC (hexane/acetone 95 : 5). Fr. 6B (337.6 mg) was submitted to another CC (toluene/MeOH 1 : 0 to
0 : 1) to give seven subfractions, Frs. 6B.1 6B.7. Further separation of Fr. 6B.4 (303.5 mg) by CC (hexane/
CH2Cl2 1 : 0 to 0 : 1) yielded 19 subfractions, Frs. 6B.4.1 6B.4.19. Fr. 6B.4.10 (17.5 mg) after PTLC
(hexane/CH2Cl2 3 : 7) resulted in the isolation of 7 (6.7 mg). Fr. 6B.4.14 (17.4 mg) furnished 8 (0.8 mg),
and a mixture of 9 and 10 (4.0 mg) after PTLC (toluene/CH2Cl2/MeOH 4 : 1 : 1). Fr. 7B (287.5 mg) was
subjected to CC (CH2Cl2/MeOH 1 : 0 0 : 1) to yield 16 subfractions, Frs. 7B.1 7B.16. Compound 11
(2.7 mg) was obtained from Fr. 7B.9 (18 mg) after PTLC (Et2O/acetone 3 : 1). Fr. 8B (84.4 mg) afforded
12 (30.5 mg) after PTLC (CH2Cl2/MeOH 9 : 1). Fr. 10B was subjected to CC (CH2Cl2/MeOH 1 : 0 to 0 : 1)
to give 15 subfractions, Frs. 10B.1 10B.15. Fr. 10B.10 (40.7) yielded 13 (6.4 mg) and 14 (20.8 mg) after
PTLC (AcOEt/MeOH 85 : 15).
Aggregatin E ( 2,3-Dihydro-3,11b-dihydroxy-3-methylnaphtho[1,2-b]oxepin-7(11bH)-one; 1). Yel-
low oil. [a] 25
D 7.23 (c 0.053, CHCl3 ). UV (MeOH): 206 (4.03), 218 (3.91), 269 (3.46). CD (c 0.02,
CHCl3 ): 381 ( 3.8), 325 ( 14.4), 300 ( 9.8), 252 (  38.3). 1H- and 13C-NMR: see the Table. ESI-MS-
152 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

MS (pos.): 265 (18, [M Li] ), 247 (100, [M Li  H2O] ). HR-ESI-MS: 265.1047 ([M Li] ,
C15H14LiO 4 ; calc. 265.1046).
Aggregatin F ( 2,3-Dihydro-3,8,11b-trihydroxy-3-methylnaphtho[1,2-b]oxepin-7(11bH)-one; 2).
Yellow oil. [a] 25
D 14.0 (c 0.04, CHCl3 ). UV (MeOH): 202 (3.44), 254 (3.04), 319 (2.77). CD (c
0.026, CHCl3 ): 369 ( 3.1), 322 ( 6.1), 291 ( 3.4), 256 ( 6.6). 1H- and 13C-NMR: see the Table. ESI-
MS-MS (pos.): 281 (100, [M Li] ), 263 (30, [M Li  H2O] ), 239 (85). HR-ESI-MS: 281.0995 ([M
Li] , C15H14LiO 5 ; calc. 281.0996).
Cytotoxicity Assay. The cytotoxic activities of 2 and 3 against UACC-62, MCF-7, NCI-ADR/RES,
786 0, NCI-H460, PC-3, OVCAR-3, HT-29, and K562 cancer cell lines, and HaCat no-cancer cell line,
were evaluated by sulforhodamine B assay [16]. The cells were distributed in 96-well plates (100 ml cells/
well) and exposed to several concentrations of 2 and 3 (0.25, 2.5, 25.0, and 250 mg/ml; 0.1% DMSO as
diluent) at 378, with 5% of CO2 , for 48 h. A 50% Cl3CCOOH soln. was added, and, after incubation for
30 min at 48, the cells were washed and dried. Cell proliferation was determined by spectrophotometric
quantification (at 540 nm) of the cellular protein content using sulforhodamine B. The experiments were
carried out in triplicate and the results were expressed as the concentration required for 50% of growth
inhibition of cell lines (IC50) in mg/ml. Doxorubicin and the solvent were used as positive and negative
control, resp. For doxorubicin, the IC50 values were 0.09  0.01 (UACC-62), 1.3  0.1 (MCF-7), 0.03 
0.00 (NCI-ADR/RES), 0.8  0.1 (PC-3), 0.03  0.00 (NCI-H460), 0.03  0.00 (786 0), 1.2  0.1
(OVCAR-3), 0.2  0.0 (HT-29), 1.3  0.1 (K562), and 0.1  0.0 (HaCat) mg/ml.

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Received July 28, 2014